牛血清白蛋白胰蛋白酶酶解产物的色谱-质谱联用分析及其三种数据库搜寻鉴定方法的比较  

Chromatography/Mass Spectrometric Analysis of BSA Digest and Comparison of Protein Identification Through Database Search with Three Different Methods

作　　者：王贤纯[1] 范春明[1] 唐新科[1] 陈平[1] 梁宋平[1] 

机构地区：[1]湖南师范大学生命科学学院,长沙410081

出　　处：《中国生物化学与分子生物学报》2004年第3期393-398,共6页Chinese Journal of Biochemistry and Molecular Biology

基　　金：湖南省自然科学基金 (No .0 1JJY2 0 2 8)资助项目~~

摘　　要：利用反相高效液相色谱 (RP HPLC)和电喷雾串联质谱 (ESI MS MS)联用技术直接对模式蛋白分子 (牛血清白蛋白 ,BSA)的胰蛋白酶酶解产物进行分离和测定 .获得的一系列BSA酶解片段的一级 (MS)和二级 (MS MS)质谱数据经分析软件处理后 ,分别在不同处理和不同参数条件下 ,用 3种不同的方法通过网上蛋白质数据库进行蛋白质搜寻鉴定 .结果显示 ,3种搜寻法都能正确地鉴定该蛋白质 ,其中以利用MS数据的肽质量指纹谱搜寻法 (PMF法 )较为快捷方便 ,但鉴定结果易受数据处理和数据库搜寻鉴定时参数设置等因素的影响 ;利用未解析MS MS数据 (rawMS MSdata)的搜寻法可在较宽的搜寻参数变化范围内获得明确的鉴定结果 ;而借助从头测序 (denovosequencing)结果的序列搜寻法 (sequencequery)则显示出更高的专一性 ,利用较少酶解片段数据就能得到稳定和明确的鉴定结果 ,搜寻参数变化的影响很小 .就酶解条件、数据处理和搜寻参数设置对蛋白质鉴定结果的影响展开详细的讨论 。The enzymatic digest of bovine serum albumin(BSA),used as a model protein, was directly analyzed by reversed phase high performance liquid chromatography (RP HPLC ) /electrospray tandem mass spectrometry (ESI MS/MS). The resulting MS and MS/MS data,after being processed with analytical software by different parameters, were used for the protein identification through database search with three different methods. All the three methods could identify the protein correctly. Peptide mass fingerprint(PMF) was the simplest and most direct method, with its results being easily influenced by parameter changes during the data processing and database search. By using raw MS/MS data, the correct identification could be obtained in wider changes of search parameters. The Sequence Query(SQ)utilizing the de novo sequencing results could make steady and definite identification based on the data of a few peptides, which was hardly influenced by the changes of main database search parameters. The effects of enzymatic digestion, data process and database search parameters on the identification of proteins were discussed in detail, which provided references for the proteomics researches.

关 键 词：色谱-质谱 牛血清白蛋白(BSA) 蛋白质数据库搜寻 鉴定方法比较 

分 类 号：Q51[生物学—生物化学] O657.63[理学—分析化学]